THE CARBOXYL-TERMINAL REGION OF FACTOR-IX IS ESSENTIAL FOR ITS SECRETION

The carboxyl-terminal region of factor M (residues 403-415) contains s everal natural mutations which result in mild to severe forms of hemop hilia B. A battery of factor IX minigene expression vectors carrying v arious mutations in the C-terminal region were constructed and studied by transient expression assay using HepG2 cells. Mutations included i n this study are Y404P, 1408N, T412N, T412S, T415G, T415S, T415L, and T415R as well. as five selected naturally occurring mutations in the r egion, R403Q, R403W, Y404H, W407R, and T412K. In comparison to the nor mal factor IX; these mutations neither significantly affected the fact or IX mRNA level nor affected the stability of the secreted factor M i n the culture medium but did decrease to various extents the intracell ular and secreted factor IX protein levels as quantified by enzyme-lin ked immunosorbent assay. T415L, T415S, and T415R showed only minor red uctions in the intracellular and minor to moderate reductions in the s ecreted factor IX levels. T415G showed only minor reduction in the int racellular factor M level but substantial reduction in the secreted le vels. R403Q, R403W, and T412S showed moderate reductions in both intra cellular and secreted factor IX levels. Y404H, Y404P, W407R, I408N, T4 12K, and T412N also showed minor to moderate reductions in the intrace llular factor IX levels but very severe reductions in the secreted fac tor M level. In one stage clotting assays, secreted factor IX mutants showed specific activities very similar to that of the normal factor I X, suggesting that the carboxyl-terminal region is neither directly in volved in the tenase complex formation required for factor X activatio n nor involved in the activation of factor IX itself. In comparison to the normal factor IX, secreted levels of factor IX mutants with mutat ions R403Q, Y404H, W407R, and T412K were also very similar to the plas ma levels reported for the hemophilia B patients carrying the same mut ations. Treatment of cells with proteasome inhibitors (ALLM and ALLN) added to the culture medium at 50 mu M resulted in drastic increases o f the intracellular mutant factor IX to the levels equivalent to that of the normal factor IX, while the secreted factor IX levels were litt le or only marginally affected. With a higher concentration of the inh ibitors (100 mu M), however, both the intracellular and secreted mutan t factor IX were fully elevated to the normal factor IX levels. Intrac ellular and secreted levels of the normal factor IX were little affect ed by the low inhibitor concentration and only marginally, if at all, at the higher concentration (less than or equal to 10%). Serine protea se inhibitors did not significantly affect intracellular and secreted factor IX levels. These results indicate that the carboxyl-terminal re gion plays a critical role in the cellular secretion of factor IX and that the mutant factor M proteins carrying specific mutations in this region are subjected to efficient elimination by the proteasome protei n degradation mechanism. Furthermore, these results strongly support t hat HepG2 cells can be utilized as a robust in vitro assay system for studying factor IX biosynthesis, well mimicking the in vivo phenomenon .